molecular interactions between the specialist herbivore ... · studies (2.0 and 2.5; reymond et...

Molecular Interactions between the Specialist HerbivoreManduca sexta (Lepidoptera, Sphingidae) and Its NaturalHost Nicotiana attenuata: V. Microarray Analysis andFurther Characterization of Large-Scale Changes inHerbivore-Induced mRNAs1

Dequan Hui, Javeed Iqbal, Katja Lehmann, Klaus Gase, Hans Peter Saluz, and Ian T. Baldwin*

Department of Molecular Ecology, Max Planck Institute for Chemical Ecology, Winzerlaer Strasse 10,D–07745 Jena, Germany (D.H., K.G., I.T.B.); and Department of Cell and Molecular Biology, Hans-Knoll-Institute for Natural Product, Beutenberg Strasse 11a, D–07745 Jena, Germany (J.I., H.P.S.)

We extend our analysis of the transcriptional reorganization that occurs when the native tobacco, Nicotiana attenuata, isattacked by Manduca sexta larvae by cloning 115 transcripts by mRNA differential display reverse transcription-polymerasechain reaction and subtractive hybridization using magnetic beads (SHMB) from the M. sexta-responsive transcriptome.These transcripts were spotted as cDNA with eight others, previously confirmed to be differentially regulated by northernanalysis on glass slide microarrays, and hybridized with Cy3- and Cy5-labeled probes derived from plants after 2, 6, 12, and24 h of continuous attack. Microarray analysis proved to be a powerful means of verifying differential expression; 73 of thecloned genes (63%) were differentially regulated (in equal proportions from differential display reverse transcription-polymerase chain reaction and SHMB procedures), and of these, 24 (32%) had similarity to known genes or putative proteins(more from SHMB). The analysis provided insights into the signaling and transcriptional basis of direct and indirect defensesused against herbivores, suggesting simultaneous activation of salicylic acid-, ethylene-, cytokinin-, WRKY-, MYB-, andoxylipin-signaling pathways and implicating terpenoid-, pathogen-, and cell wall-related transcripts in defense responses.These defense responses require resources that could be made available by decreases in four photosynthetic-relatedtranscripts, increases in transcripts associated with protein and nucleotide turnover, and increases in transcripts associatedwith carbohydrate metabolism. This putative up-regulation of defense-associated and down-regulation of growth-associatedtranscripts occur against a backdrop of altered transcripts for RNA-binding proteins, putative ATP/ADP translocators,chaperonins, histones, and water channel proteins, responses consistent with a major metabolic reconfiguration thatunderscores the complexity of response to herbivore attack.

Plants are known to exhibit large phenotypicchanges when confronted with various abiotic andbiotic insults, and these changes are thought to in-crease plant fitness if the insults continue over time.The mechanisms responsible for these examples ofadaptive phenotypic plasticity are largely unknown,but analyses of responses with Arabidopsis clearlyindicate that a large proportion of the transcriptomeis involved (Maleck et al., 2000; Schenk et al., 2000;Sasaki et al., 2001). Transcriptional responses to en-vironmental stresses are exceptionally complicatedbecause they require a deep understanding of boththe environmental parameters that determine aplant’s fitness and metabolism sensu lato. Plant re-sponses to herbivore attack, for example, involve theactivation of direct and indirect defenses and toler-

ance responses, which can be specific to the attackingherbivore, as has been demonstrated in the Nicotianaattenuata-Manduca sexta plant herbivore system(Baldwin, 2001; Baldwin et al., 2001).

When attacked by the nicotine-tolerant specialistM. sexta, N. attenuata “recognizes” the attack, as evi-denced by alterations in a number of its wound- andjasmonate (JA)-elicited responses. The induced JAlevels that are normally proportional to the amountof mechanical wounding erupt into a JA burst thatincreases concentrations 2 to 10 times that of wound-induced levels and is propagated throughout thedamaged leaf ahead of the rapidly foraging herbivore(Schittko et al., 2000; Ziegler et al., 2001). Woundingand JA elicitation caused by wounding do not pro-voke ethylene emissions, but M. sexta attack pro-duces a rapid ethylene burst, which is sustained dur-ing larval feeding (Kahl et al., 2000). The ethyleneburst suppresses the wound- and JA-induced accu-mulation of nicotine biosynthetic genes, NaPMT1 and2, and the associated nicotine accumulation (Winzand Baldwin, 2001). The ethylene burst does not,

1 This work was supported by the Max Planck Gesellschaft.* Corresponding author; e-mail [email protected]; fax

49 – 0 –3641–571102.Article, publication date, and citation information can be found

at www.plantphysiol.org/cgi/doi/10.1104/pp.102.018176.

Plant Physiology, April 2003, Vol. 131, pp. 1877–1893, www.plantphysiol.org © 2003 American Society of Plant Biologists 1877

however, suppress the release of volatile organiccompounds, which function as powerful indirect de-fenses in nature by attracting a generalist predator tothe feeding herbivore (Kessler and Baldwin, 2001)and are also elicited by larval feeding but not bymechanical wounding (Halitschke et al., 2000; Kahl etal., 2000). In summary, at a phenotypic level of anal-ysis, M. sexta attack of N. attenuata results in a down-regulation of a major direct defense, nicotine, whichis demonstrably effective against mammalian herbi-vores, and an up-regulation of an indirect defense,the release of predator-attracting volatiles, which inturn is demonstrably effective against insect herbi-vores. Because nicotine can be sequestered by M.sexta for its own defense against parasitoids, the M.sexta-induced changes likely represent an adaptivetailoring of N. attenuata’s wound response.

To understand the transcriptional basis of these M.sexta-induced changes in defense strategies, we useddifferential display reverse transcription (DDRT)-PCR with one arbitrary primer to gain an unbiasedview of approximately 5% of the M. sexta-inducedtranscriptome. This analysis identified 53 individualsequences, of which 49 were detectable on RNA gelblots, and differential expression was verified for 27(Hermsmeier et al., 2001). Here, we provide a secondinstallment in the analysis of the M. sexta-alteredtranscriptome by continuing the DDRT-PCR analysiswith six additional random primers, each with 10anchor primers. Because DDRT-PCR provides se-quence from the 3�-untranslated region of genes,which tends to be highly gene- and species-specificand, therefore, diminishes the probability of findinghomology with genes of similar function in the data

bases, a subtractive hybridization using magneticbeads (SHMB; Sharma et al., 1993) was used to com-plement the analysis with sequences more likely tooriginate from the open reading frame (ORF) of M.sexta-induced N. attenuata genes. All genes cloned byDDRT-PCR and SHMB (53 from Hermsmeier et al.,2001; 115 from this study; and 10 “control” genes)were spotted as cDNAs on glass slide microarrays(see Table I). We examined the transcriptionalchanges of the cloned transcripts by hybridizing themicroarrays with fluorescently labeled transcriptsfrom plants massively attacked by M. sexta larvae for2, 6, 12, and 24 h and provide full-length sequences oftwo genes that catalyze the early and final steps inthe biosynthesis of terpenoid-derived defense metab-olites: 3-hydroxy-3-methylglutaryl CoA reductase(HMGR) and 5-epi-aristolochene synthase (EAS). Theanalysis highlights the extent to which metabolism isreconfigured during herbivore attack.

RESULTS

We cloned a total of 115 transcripts by DDRT-PCRand SHMB from N. attenuata plants under continuousattack from 20 first instar M. sexta larvae for 24 h. ThecDNAs of all cloned transcripts, in addition to eightpreviously characterized M. sexta-induced genes,were spotted on Lys-coated glass slide microarraysand hybridized with Cy3- and Cy5-labeled mRNAprobes isolated from N. attenuata plants subjected toattack from the same number of larvae but harvestedat 2, 6, 12, and 24 h after the start of the attack to fullycharacterize the response.

Table I. Genes cloned by DDRT-PCR (DD/arbitrary primer no.) and subtractive hybridization with magnetic beads (SHMB) that exhibitednonsignificant (between 0.5 and 1.50) expression ratios in the microarray analysis and had similarity to genes in the databases

Genes are listed in order of decreasing E value from BLAST queries. Expression patterns are defined as: gradual (Type Ia) or abrupt (Type Ib)increases; initial decreases followed by either steady (Type IIa) or abrupt increases (Type IIb); an initial increase followed by a decrease (TypeIII); an increase, a decrease, and finally an increase (Type IV; both peaks are given if equal) as plants were continuously attacked by Manducasexta larvae over 24 h. Down-regulated genes have the opposite patterns.

Clone Length MethodPeakat h

ExpressionRatio

TypeAccession

No.Sequence Similarity E Value

bp

DH017 1028 SHMB 24 1.33 IIb CA591807 Matrix attachment regions-binding protein(MARBPF; AB059832)

0.00

DH108 453 SHMB 12 0.80 III CA591793 PSII (NtPII10; X70088) 0.00RE322 590 DD/R5 24 1.25 Ia CA591771 Transcription factor NtWRKY2 (AB020590) 1 � 10–120DH123 371 SHMB 24 1.21 IIa CA591796 Cytosolic glyceraldehyde-3-phosphophate

dehydrogenase (GapC; M14419)2 � 10–94

RE283 184 DD/R5 12 0.63 III CA591769 PSII oxygen-evolving complex,23-kD polypeptide (X58910)

1 � 10–73

RD161 234 DD/R4 12 1.47 III CA591756 Actin gene Sfa 15B (X03076) 1 � 10–47RB142 107 DD/R2 24 1.13 IIb CA591717 25S ribosomal RNA gene (X13557) 1 � 10–43RF064 161 DD/R6 24 1.20 IIb CA591783 26S ribosomal RNA gene (AF479172) 2 � 10–43RD131 190 DD/R4 12 1.30 III CA591754 Signal recognition particle 7S RNA (Z29099) 3 � 10–35DH270 307 SHMB 6, 24 1.20, 1.23 IV CA591818 Type 2 metallothionein-like protein (U35225) 6 � 10–31RN021 389 DD/R14 24 1.43 Ia CA591773 Beta-alanine synthase (Y19104) 3 � 10–30RN032 337 DD/R14 12 0.54 III CA591774 Polypeptide of PSII (X85038) 1 � 10–17RC231 276 DD/R3 12 1.23 III CA591748 myb1 gene (AF248962) 8 � 10–17

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1878 Plant Physiol. Vol. 131, 2003

Utility and Limitations of the Microarrays

Due to variation in the spot placement and shapeon these Lys slides, background corrections for eachspot were performed manually, and not all spotscould be used. We arbitrarily defined cDNAs withmean (of a maximum of eight replicate spots, range5–8) expression ratios of �0.5 or �1.50 as being dif-ferentially expressed (down- and up-regulated, re-spectively). These thresholds are higher than thoseused in the companion paper (Halitschke et al., 2003;�0.75 or �1.25) because sample size constraints didnot allow for the use of the statistical criteria indetermining differential expression that were used inthe companion paper, but lower than those of otherstudies (2.0 and 2.5; Reymond et al., 2000) that usedonly a spot per cDNA and, hence, had no means ofdetermining within-array variance.

The cDNAs from eight N. attenuata genes knownto respond to herbivore attack, which had beenpreviously analyzed by RNA blots, were used tomonitor the entire experimental process and de-termine whether the microarrays provided thesame results as the northern-blot analysis. Previouswork (Halitschke et al., 2001; Hermsmeier et al.,2001; Schittko et al., 2001; Winz and Baldwin, 2001;

Ziegler et al., 2001; Glawe et al., 2003) had estab-lished that transcripts of Thr deaminase, proteinaseinhibitor (PI), allene oxide synthase (AOS), alpha-dioxygenase (�-DIOX), hydroperoxide lyase (HPL),and putrescine N-methyl transferase (PMT) werestrongly up-regulated, whereas transcripts of ribu-lose-1,5-biphosphate carboxylase (RuBPCase) weredown-regulated after M. sexta larvae attack, com-pared with unattacked plants. Microarray analysis(Figs. 1 and 2) confirmed these results, thereby es-tablishing the utility of the procedure for this sys-tem. It should be noted that although some of thesecontrol genes tended to be strongly regulated inboth the array analysis and previous northern anal-yses, others, such as AOS, which are strongly regu-lated on northern blots within 30 min of elicitation(Ziegler et al., 2001), were found to be significantlyregulated only at the 24-h harvest with the microar-ray (Fig. 1), suggesting that the arbitrary thresholdsmay exclude differentially regulated genes. Hence,to avoid disposing of potentially valuable informa-tion, we provide a list of genes with significantsimilarity to known genes in the database that werenot differentially regulated by the established crite-ria (Table I).

Figure 1. Mean (�SD) expression ratios from microarrays with eight replicate cDNA spots of partial sequences of N.attenuata Thr deaminase (TD; note break in y axis and that only � SD are shown), proteinase inhibitor (PI), PMT, andRuBPCase small subunit genes hybridized with fluorescently labeled probes derived from M. sexta-attacked or control N.attenuata plants (harvested 2, 6, 12, or 24 h after the start of attack). Shaded area represents arbitrarily defined zone ofnonsignificant changes in expression.

Microarray Analysis of Insect-Induced Transcript Accumulation

Plant Physiol. Vol. 131, 2003 1879

Transcriptional changes, measured on the microar-rays for each gene during the 24 h of continuous M.sexta larvae attack, were categorized as being: grad-ual (Type Ia) or abrupt (Type Ib) increases over the24-h time course; an initial decrease followed byeither steady (Type IIa) or abrupt increases (TypeIIb); an initial increase followed by a decrease (TypeIII); an increase, followed by a decrease and an in-crease (Type IV). Down-regulated genes were simi-larly classified (Tables I and II). It should be notedthat significant expression ratios (or lack thereof) inone of the four harvests with these arrays should beviewed as an indication of differential expression, anindication that should be confirmed with additionalnorthern-blot analysis.

Comparison of DDRT-PCR and SHMB

From six rounds of DDRT-PCR with six differentarbitrary primers (each with 10 anchor primers), wecloned and sequenced 84 different transcripts thatranged in size from 107 to 535 bp. Of these, 46 tran-scripts (60%), including 17 with similarity to knowngenes, had an expression ratio of greater than orequal to 1.50, predominantly at the 24-h harvest. Twotranscripts had an expression ratio � 0.5 at 12 and

24 h (Table II). Nine expressed sequence tags (ESTs)with significant similarity to known genes (Table I)and 24 ESTs with no significant similarity to knowngenes did not show differential expression as de-fined. Hence, we could not confirm differential ex-pression in 40% of the transcripts identified byDDRT-PCR, and 73% had no similarity to genes inthe databases (data not shown).

From SHMB, we sequenced 33 of 60 clones (fromone-fourth of the final ligation volume, presumablyrepresenting 25% of induced transcripts) that rangedin size from 137 to 937 bp. Twenty-two (73%) of thesetranscripts, which included 15 with similarity toknown genes (Table II), showed expression ratios �1.50 predominantly at the 24-h harvest. Two tran-scripts (Table II), including one with similarity to 25Sribosomal RNA gene, had an expression ratio smallerthan 0.5 at the 2-h harvest. In addition, five tran-scripts with significant similarity to known genes(Table I) and four transcripts with no significant sim-ilarity did not show any differential expression.Hence, we could not confirm differential expressionin 27% of the sequenced transcripts identified bySHMB, and 44% had no similarity to genes in thedatabases.

Figure 2. Mean (�SD) expression ratios from microarrays with eight replicate cDNA spots with partial sequences of genesmediating N. attenuata’s oxylipin cascade (lipoxygenase [LOX], HPL, AOS, and �-DIOX) hybridized with fluorescentlylabeled probes derived from M. sexta-attacked or control N. attenuata plants (harvested 2, 6, 12, or 24 h after the start ofattack). Shaded area represents arbitrarily defined zone of nonsignificant changes in expression.

Hui et al.


Overall, a total of 73 transcripts (63% of the clonedgenes) were confirmed to be differentially expressed(down- or up-regulated) with the microarrays (Ta-bles II and III). Of these, 32% of the transcripts ex-hibited a Type Ia, 8% a Type Ib, 41% a Type IIa, 7%a Type IIb, 8% a Type III, and 4% a Type IV expres-sion pattern over the time course of the experiment.Twenty-four differentially expressed transcripts(32%) had similarity to known genes or putativeproteins. The 14 ESTs with significant sequence sim-ilarity to known genes that could not be confirmed tobe differentially expressed (Table I) tended to bedown-regulated at the 2-, 6-, and 12-h harvests,

whereas exhibiting modest up-regulation (expressionratios between �1.0 and �1.5) at the 24-h harvest. Insummary, the proportion of transcripts cloned byDDRT-PCR and SHMB that could be confirmed to bedifferentially expressed was similar between the twoprocedures, but as expected (Appel et al., 1999),SHMB produced a greater proportion of clones withsequences with significant similarity to genes in thedatabases. These genes could be crudely categorizedas being involved in oxylipin signaling, transcrip-tional regulation, terpenoid biosynthesis, antimicro-bial defense, and the remodeling of cell walls andmetabolism.

Table II. Genes cloned by DDRT-PCR (DD/arbitrary primer no.) and subtractive hybridization with magnetic beads (SHMB) that exhibitedsignificant (�0.5 and �1.50) expression ratios in the microarray analysis and had similarity to genes in the databases

Genes are listed in order of decreasing E value from the BLAST queries. Expression patterns are defined as: gradual (Type Ia) or abrupt (TypeIb) increases; initial decreases followed by either steady (Type IIa) or abrupt increases (Type IIb); an initial increase followed by a decrease (TypeIII); an increase, a decrease, and finally an increase (Type I;: both peaks are given if equal) as plants were continuously attacked by M. sexta larvaeover 24 h. Down-regulated genes have the opposite patterns.

Clone Length MethodPeakat h

ExpressionRatio

TypeAccession

No.Sequence Similarity E Value

bp

DH120 394 SHMB 24 2.38 IIa AF542543 3-Hydroxy-3-methylglutaryl-CoA reductase (X63649) 0DH164 452 SHMB 24 1.58 IIa AF542544 5-Epi-aristolochene synthase mRNA (AF272244) 1 � 10–168DH114 372 SHMB 2 0.40 Ia CA591794 26S Ribosomal RNA gene spacer (Y08427) 1 � 10–156DH083 607 SHMB 6, 24 1.66, 168 IV CA591811 Tumor-related protein (D26464) 1 � 10–137RN254 487 DD/R14 24 1.90 Ia CA591779 Ubiquitin carrier protein (Ubc-E2) mRNA (L23762) 1 � 10–120DH099 482 SHMB 24 3.34 Ia CA591812 Basic pathogenesis-related protein (PR1; X14065) 1 � 10–115RN161 248 DD/R14 24 2.11 Ia CA591777 Transformer-2-like SR ribonucleoprotein (RNP;

Y09506)1 � 10–113

DH104 594 SHMB 24 1.66 IIa CA591822 Thiazole biosynthetic enzyme precursor (NM124858)

1 � 10–86

DH054 224 SHMB 24 2.04 Ia CA591790 Sulfite reductase (AB010717) 2 � 10–73RB271 484 DD/R2 12 2.33 IV CA591719 Xyloglucan endo-transglycosylase (X82685) 2 � 10–77RB521 484 DD/R2 24 3.44 IV CA591731 Xyloglucan endo-transglycosylase (X82685) 1 � 10–75RB061 230 DD/R2 24 2.88 Ia CA591715 13-Lipoxygenase clone H3 (X96406) 2 � 10–72RC191 233 DD/R3 24 2.70 Ia CA591745 ant Gene for ATP/ADP translocator (X62123) 8 � 10–72RB131 427 DD/R2 24 1.51 IIb CA591716 Allene oxide synthase (AJ457080) 3 � 10–65DH162 461 SHMB 24 2.21 Ia CA591801 RNA-binding Gly-rich protein (RGP-1a; D16204) 1 � 10–61RF113 561 DD/R6 24 2.31 IIa CA591788 Cytokinin-induced (cig2) mRNA (AB031321) 9 � 10–57RB493 231 DD/R2 2 1.91 IIa CA591728 (Zymonaonas mobilis) rrnB operon (AF088897) 1 � 10–55DH283 353 SHMB 12 1.64 III CA591810 Chaperonin 60 (X70868) 2 � 10–55DH124 586 SHMB 24 1.63 IIa CA591814 (Nicotiana tabacum) GTP-binding protein (Ran-A1)

mRNA (L16767)5 � 10–44

RF071 255 DD/R6 12 0.50 IIa CA591784 Ribulose-1,5-biphosphate carboxylase small subunit(M32420)

1 � 10–33

RB012 289 DD/R2 24 1.51 IIa CA591712 Alpha-amylase (amy21) mRNA (M81682) 1 � 10–27DH219 341 SHMB 24 1.76 Ia CA591804 Histone H3 (PcH3–20) gene (M77494) 1 � 10–18DH193 359 SHMB 24 4.96 Ib CA591816 Major intrinsic protein (MIP) 2 (Y18312) 7 � 10–18DH182 627 SHMB 24 3.65 Ib CA591802 Ser carboxypeptidase cp-b (AJ 251970) 2 � 10–16RB481 535 DD/R2 24 1.79 Ia CA591726 (Arabidopsis) putative protein (MN_127327) 1 � 10–11DH138 295 SHMB 24 2.09 IIa CA591799 60S ribosomal protein (NM_114851) 2 � 10–11RB304 425 DD/R2 24 1.67 IIa CA591720 (Lotus japonicus) genomic DNA, chromosome 6

(AP004526)2 � 10–09

RE234 326 DD/R5 2 1.69 IIa CA591767 Plastidic aldolase-like protein (AB027001) 3 � 10–08RE112 345 DD/R5 24 4.38 Ia CA591761 Ser protease sbt4b (AJ006480) 6 � 10–06RC144 342 DD/R3 12 1.83 IIb CA591742 VFNT Pto locus (AF220603) 7 � 10–06DH061 399 SHMB 24 1.70 Ia CA591791 (Arabidopsis) putative protein, mRNA (MN_117913) 9 � 10–06DH126 302 SHMB 12 0.43 IIa CA591815 ATP-binding cassette (ABC) transporter protein 1

(MN_125882)1 � 10–3

RC095 496 DD/R3 24 2.41 Ia CA591780 Ubiquitin carrier protein (Ubc) mRNA (L23762) 0.040



Oxylipin Signaling and Transcriptional Regulation

Two transcripts identified by DDRT-PCR (RB061and RB131) with similarity to potato (Solanum tubero-sum) LOX (Royo et al., 1996) and tomato (Lycopersiconesculentum) AOS (Sivasankar et al., 2000), respec-tively, as well as HPL (J. Zeigler and R. Halitschke,unpublished data) were strongly up-regulated by M.sexta attack (Fig. 2; Table II). The 13-lipoxygenasecatalyzes the dioxygenation of fatty acids with a 1,4-pentadiene structure to produce, among other prod-ucts, 13-hydroperoxy linoleic acid, which is a sub-strate for AOS and HPL, initiating the biosynthesis ofJA or volatile C6 compounds.

Interestingly, the 427-bp fragment of AOS cloned byDDRT-PCR (RB131) had greater similarity (86%) to thetomato gene than to the NaAOS previously clonedfrom N. attenuata (80%). Southern-blot analysis ofN. attenuata genomic DNA digested with HindIII andXbaI, for which one recognition site per enzyme existswithin the NaAOS ORF, revealed a complex bandingpattern consistent with the existence of two genescoding for AOS in the N. attenuata genome (Ziegler etal., 2001). NaAOS originated from a screen of a cDNAlibrary constructed from equal quantities of M. sexta-attacked N. attenuata leaves from seven genotypes,including the genotype used in this study. Althoughthe microarray revealed that both NaAOS (Fig. 2) andRB131 had Type IIa expression patterns, the sequencedifferences between RB131 and NaAOS are likely toosubstantial to reflect allelic differences, and it is pos-sible that RB131 represents a fragment from the sec-ond aos in the N. attenuata genome.

Although transcripts of LOX, AOS, and HPL exhib-ited coordinated Type I or II patterns of expression(Fig. 2), �-DIOX, which catalyzes the alpha-oxidationof fatty acids to hydroperoxy fatty acids and may beinvolved in signal generation (Sanz et al., 1998; Ham-berg et al., 1999), exhibited a Type IV pattern ofexpression (Fig. 2). Prior work with �-DIOX demon-strated that transcripts increased rapidly in leavesattacked by M. sexta larvae and that fatty acid aminoacid conjugates (FACs) in the oral secretions wereresponsible for up-regulating the wound-induced in-crease (Halitschke et al., 2001; Schittko et al., 2001).The type IV expression patterns are likely to reflectthe interplay between wound-induced and oralsecretion-mediated increases during massive cater-pillar attack.

The clones coding putative transcription factors(RE322 encoding 590 bp of a WRKY transcriptionfactor and RC231 encoding 276 bp of a putative MYBtranscription factor) were found to be down-regulated both on DDRT-PCR display gels and at the2- and 6-h harvests with the microarray. However, inlater harvests (12 h for RE322 and 24 h for RC231),both tended to be up-regulated but not with expres-sion ratios above the arbitrarily defined threshold of1.5 (Table I). WRKY transcription factors occur inlarge gene families and are known to regulate a

plethora of different genes, including pathogen- andwound-induced gene expression, by binding toW-box promoter elements in a variety of plant spe-cies (Eulgem et al., 2000). WRKY transcription factorsare known to bind to W-box elements in PR1 genes (ahomolog of which was cloned by SHMB; Table II)and regulate their expression after salicylic acid (SA)induction and pathogen elicitation (Rushton et al.,1996). Although many WRKY factors are known to bewound induced, few if any studies have found themto be induced by herbivore attack or JAs. Similarly,the expression of MYB transcription factors areknown to be induced by tobacco mosaic virus andbacterial pathogen infection and SA treatment, withsubsequent induction of PR genes (Yang and Klessig,1996), and are generally involved in the regulation ofphenylpropanoid metabolism, cell shape, and hor-mone signal transduction (Martin and PazAres,1997). More recently, a novel MYB was found in rice(Oryza sativa) that is JA inducible (Lee et al., 2001).

Terpenoid Biosynthesis

The SHMB analysis provided the DH120 andDH164 clones that code for HMGR (Genschik et al.,1992) and EAS (Mandujano-Chavez et al., 2000;Bohlmann et al., 2002), respectively. The enzymescatalyze the early and final steps in terpenoid bio-synthesis, in which acetyl CoA and acetoacetyl CoAare converted to HMG CoA by 3-hydroxy-3-methylglutaryl CoA synthase, reduced to mevalonateby HMGR, and subsequently converted to isoprenoidpyrophosphate, the universal precursor for isopre-noids. EAS is a terpenoid synthase that catalyzes thecyclization of farnesyl diphosphate to the sesquiter-penoid (5-epi-aristolochene) for the subsequent for-mation of bicyclic sesquiterpenoid phytoalexin cap-sidiol (Bohlmann et al., 2002). The expression of bothgenes after M. sexta attack was highly coordinated:Both were initially down-regulated with subsequentincreases in expression as herbivore attack pro-ceeded, but the expression ratios for HMGR weregreater than that of EAS at the 24-h harvests (Fig. 3).

Because HMGR was one of the first transcriptsshown to be differentially regulated in potato afterattack by M. sexta larvae (Korth and Dixon, 1997) andis known to exist in a gene family and thought to playan essential role in regulating substrate flux into thecytosolic pathway of terpenoid biosynthesis (Korth etal., 2000), we amplified a 2,000-bp HGMR cDNAfrom N. attenuata by PCR with primer HMGf1 (5�-CGGCAATCTTACCGGTGAAA) derived from theHMGR cDNA sequence of Nicotiana sylvestris andprimer HMGr1 (5�-TGAGATAGCTGACATGAGGG)derived from clone DH120. The DNA sequence con-tains an ORF of 1,812 nucleotides (AF542543) andencodes 604 amino acids with a calculated molecularmass of 65,125 D. The amino acid sequence showed95% homology to N. sylvestris HMGR, 87% to pepper

Hui et al.


(Capsicum annuum), and 85% to N. tabacum (Fig. 4).The deduced protein consists of two transmembranedomains in the N-terminal region and a C-terminalcatalytic domain of 349 amino acid residues contain-ing the three highly conserved signatures located inthe center of the catalytic domain, in a Gly-rich re-gion, and in a region containing a His residuethought to be essential for catalytic activity (Fig. 4).

Prior work on N. attenuata identified three new cop-ies of EAS (NaEAS-12, -34, and -37) by screening acDNA library of N. attenuata leaves from plants orig-inating from seven geographically distinct popula-tions, including the genotype used in this study, all ofwhich had been attacked by M. sexta larvae (Bohl-mann et al., 2002). To determine whether the EAScloned by SHMB was the same as one of the previ-ously cloned ones, a 1,950-bp cDNA (NaEASutah;AF542544) was generated by PCR with primer EPIf1(5�-AATACACTCATCTTTAATTAG) derived fromthe N. tabacum ESA cDNA sequence and primerEPIr1 (5�-CACTAGCTTCAAGAATTTTAG) derivedfrom clone DH164. The sequence contains an ORF of1,647 nucleotides and encodes 548 amino acids of acalculated molecular mass of 62,895 D (Fig. 5), whichshowed 92% similarity to N. tabacum gene and 92% to93% similarity to the previously cloned N. attenuatagenes. Structurally, the enzyme is organized into twodomains and there are two Mg2�-binding sites lo-

cated in the C-terminal domain (Starks et al., 1997;Fig. 5). Elicitor-induced EASs from N. tabacum areknown to occur in small gene families (Facchini andChappell, 1992), and recently an EAS from corn (Zeamays; stc1; (Shen et al., 2000) has been shown to beinduced by volicitin, the fatty acid-amino acid con-jugate found in Spodoptera exigua oral secretions thatelicits the release of volatiles in this species.

Cell Wall Remodeling

A total of 20 sequences from the DDRT-PCR anal-ysis with arbitrary primer 2 could be assembled intotwo contigs of the same length (484 bp), representingclones RB271 and RB521, which had 88.4% similarityto tomato XG endo-transglycosylase/hydrolyase(tXET-B1; Arrowsmith and Desilva, 1995; renamedXTH by Yokoyama and Nishitani, 2002). Both exhib-ited a Type IV expression pattern (Fig. 3). XTHs arethought to function in the cleavage and concomitanttransfer of XG molecules into plant cell walls bytransglycosylation and may mediate the loosening ofcell walls during growth (Emons and Mulder, 2000).

Antimicrobial-Associated Genes

SHMB provided three clones that are implicated inpathogen resistance. One of these, DH099, has simi-

Figure 3. Mean (�SD) expression ratios from microarrays with eight replicate cDNA spots with partial sequences of N.attenuata xyloglucan (XG) endo-transglycosylase (XTH1), basic pathogenesis-related protein (PR1), HMGR, and EAS geneshybridized with fluorescently labeled probes derived from M. sexta-attacked or control N. attenuata plants (harvested 2, 6,12, or 24 h after the start of attack). Shaded area represents arbitrarily defined zone of nonsignificant changes in expression.



larity to the basic-type pathogenesis-related proteinPR1 of N. tabacum (Payne et al., 1989), and the mi-croarray analysis demonstrated that it was stronglyup-regulated by M. sexta attack (Fig. 3). Basic PR1sare intracellular proteins associated with viral, fun-gal, and bacterial infections and are strongly elicitedby ethylene (Kitajima and Sato, 1999). Therefore, theobserved PR1 response is expected, given that M.sexta attack is known to elicit an ethylene burst in N.attenuata (Kahl et al., 2000). SHMB also providedDH083, which had similarity to genetic tumor-related cDNA in an interspecific hybrid (F1) betweenNicotiana glauca and Nicotiana langsdorffii (Fujita et al.,1994) and DH270, which had similarity to a Nicotianaplumbaginifolia type 2 metallothtionein-like protein-encoding gene (Table I). Transcripts of both exhibiteda Type IV expression pattern; however, DH270’s in-creases were never higher than a 1.23 expressionratio (Table I), whereas DH083 attained 1.66 and 1.68at the 6- and 24-h harvests (Table II). Some type 2metallothioneins are thought to function as potentmetal chelators (Giritch et al., 1998), but others playroles in different cell death pathways, including senes-cence and the hypersensitive response (HR) after

pathogen attack (Butt et al., 1998). Pathogen recogni-tion is implicated in the up-regulation of clone RC144,which has similarity to a putative pto gene (D.T.Lavelle, G.E.D. Oldroyd, D. Dalhbeck, B.J. Staskawicz,and R.W. Michelmore, unpublished data). The ptogene complex codes for protein kinases that mediateresistance against Pseudomonas syringae pv tomato in-fections and is correlated with HR (Loh and Martin,1995). Clone DH126 (Table I) has similarity with aputative Arabidopsis ABC transporter (C.D. Town, B.JHaas, R. Maiti, L.I. Hannick, A.P. Chan, C.M. Ronning,R.K. Smith Jr, C.Y. Yu, J.R. Wortman, O. White et al.,unpublished data). The superfamily of ABC trans-porter genes code for ATP-driven membrane associ-ated efflux pumps that export a range of cytotoxiccompounds. In N. tabacum, transcripts for a ABCtransporter are reported to be JA elicited (Sasabe et al.,2002) and in tomato, an ABC transporter called pti3 isknown to interact with pto in two-hybrid screens.

Remodeling of Metabolism

The remaining clones with similarity to knowngenes reflect the extent to which metabolism (sensu

Figure 4. Alignment of deduced amino acid sequences of N. attenuata HMGR (AF542543), N. sylvestris (S24760), N.tabacum (AAB87727), and pepper (Q9XEL8). Missing amino acids are indicated by dashes, and different amino acids areindicated by black shading. A transmembrane domain with two segments located in the N-terminal region and a 349-aminoacid residue catalytic domain containing three highly conserved signatures, located in the center of the catalytic domain (1),in a Gly-rich region (2), and in a region containing a His residue (3), are indicated by boxes and gray shading.

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lato) is reorganized, presumably to make resourcesavailable for regrowth-, repair-, and defense-relatedprocesses after massive herbivore attack.

Five photosynthesis-related genes were identifiedby DDRT-PCR and SHMB, but only one RF071; TableI) was found to be significantly down-regulated byM. sexta attack (Fig. 1). RF071 codes for a transcrip-tionally active pseudogene of the small subunit geneof RuBPCase complex, which catalyzes the photosyn-thetic fixation of CO2 through the Calvin cycle (Onealet al., 1987). The down-regulation of this gene com-plements that of the functional RuBPCase small sub-unit of Rubisco gene found in the Hermsmeier et al.(2001) study. Three photosynthetic-related tran-scripts (DH108, PSII; NtPII10, Zhou et al., 1993;RE283, 23-kD polypeptide of PSII oxygen-evolvingcomplex, Hua et al., 1991; and RN032, 6.1-kDpolypeptide of PSII, Lorkovic et al., 1995) all tendedto be down-regulated with the lowest expression ra-tio at the 12-h harvest (Table I).

The down-regulation of genes related to photosyn-thesis may allow attacked plants to reinvest re-sources into other processes; a similar reinvestmentfunction may be played by RN021 and DH182, bothof which could allow for the recovery of amino acidsinvested in pyrimidine and protein synthesis, respec-tively. RN021, which has similarity to transcripts forbeta-Ala synthase (C. Chevalier, J. Joubes, J. Petit,and P. Raymond, unpublished data), an enzyme thatcatalyzes the third and final step of pyrimidine ca-tabolism to produce beta-Ala, was slightly up-regulated. DH182 was strongly up-regulated (TableII) and exhibits homology to barley (Hordeum vulgare)and Arabidopsis Ser carboxypeptidase (Dal Degan etal., 1994.). A member of this gene family, Ser CPII(BRS1), plays a regulatory role in the brassinosteroid(BR) signaling (Li et al., 2001), which is mediated byBR1 receptor in BR-insensitive (BRI1) mutants. Intomato, type I Ser CPs are among the “late wound-inducible” genes. Elicited by JAs and systemin, they

Figure 5. Alignment of deduced amino acid sequences of EAS from different genotypes of N. attenuata (EASutah[AF542544], EAS12 [AF484123], EAS34 [AF484124], and EAS37 [AF484124]) and N. tabacum (5EAS). Amino aciddifferences are indicated by black shading. The gene consists of two domains: domain 1 (enclosed in box with a dotted line),which bisects domain 2 into two parts (both enclosed in boxes with a solid lines). Two conserved Mg2�-binding sites locatedin C-terminal portion of domain 2 are indicated by gray shading.



may be involved in general protein turnover ratherthan signaling (Moura et al., 2001). Clone RE112, withsimilarity to a Ser protease of the subtilase genefamily (Meichtry et al., 1999), could be playing a rolein signaling because related members of this familyare thought to process the wound hormone systemin.Two clones, RN254 and RC095, with sequence simi-larity to the tomato ubiquitin carrier protein (ubc) oralternatively, ubiquitin-conjugating enzyme (E2;D.M. Bird and M.A. Wilson, unpublished data),which is responsible for recognizing and tagging ap-propriate targets for the main non-lysosomal routefor intracellular protein degradation in response tostress (Jesenberger and Jentsch, 2002), were found tobe gradually up-regulated by herbivore attack. Theubiquitin protein-conjugating system plays a pivotalrole in the ubiquitin-dependent proteosome pathwayin the regulation of apoptosis or programmed celldeath, the central process of the HR that is an impor-tant means of limiting the spread of pathogens inplants. However, because an HR is not observed afterM. sexta attack, E2 is more likely involved in selectiveprotein turnover.

Although alterations in these transcripts may di-rectly or indirectly help a plant to meet its amino acidor more generally, nitrogen requirements for the nec-essary metabolic reconfiguration, Glc demands mightbe met by the hydrolysis of starch and oligosaccha-rides, as is suggested by the up-regulation of RB012, atranscript homologous to alpha-amylase (K. Gausingand T. D. Kreiberg, unpublished data). Alpha-amylaseis normally GA inducible, but in this case it is gradu-ally up-regulated during a massive herbivore attack.The expression of DH123, a 371-bp fragment withsimilarity to the cytosolic glyceraldehyde-3-phos-phophate dehydrogenase (Shih et al., 1986) tended tobe down-regulated (at the 2-, 6-, and 12-h harvests).Glyceraldehyde-3-phosphophate dehydrogenase par-ticipates in carbohydrate metabolism and is known tobe elicited by SA and pathogen infection in potato(Laxalt et al., 1996) and by anaerobic conditions incorn (Manjunath and Sachs, 1997). RE234, which cor-responds to plastidic aldolase (aldP; Yamada et al.,2000), was up-regulated shortly after the start ofherbivore attack. aldP is involved in photosyntheticcarbon reduction and catalyzes the synthesis of Fru-1,6 bis-phosphate from d-glyceraldehyde-3-phos-phate and dihydroxyacetone phosphate. AldP tran-scripts are up-regulated after salt stress in variousNicotiana spp. (Yamada et al., 2000), and smallchanges in AldP activity by antisense-mediated genesilencing have dramatic effects on photosynthesis(Haake et al., 1998). The changes in energy metabo-lism elicited by herbivore attack will likely requireincreases in the exchange of ADP and ATP betweenthe cytosol and mitochondria. RC191 corresponds topotato adenine nucleotide translocase (ANT) gene forADP/ATP translocator (Emmermann et al., 1991)and is strongly up-regulated in herbivore-attacked

plants (Table II). ANT is a translocator protein essen-tial for the formation of the mitochondria permeabil-ity transition pore and is the most abundant proteinof the inner mitochondrial membrane.

Transcripts for another mitochondria protein,DH283, a chaperonin of the 60-kD heat shock protein(Hsp) family (Prasad et al., 1990; Tsugeki et al., 1992),are thought to be important in the folding and as-sembly of multimeric proteins in the mitochondria.This is the second Hsp cloned from N. attenuata thatis elicited by M. sexta attack. The first was AW191822,which has similarity to luminal-binding proteins andwas also up-regulated by herbivore attack (Herms-meier et al., 2001). Other researchers have examinedthe production of Hsp in N. attenuata and foundevidence for systemic elicitation by methyl JA of anHsp70 and smaller Hsps (16–23 kD; Hamilton andColeman, 2001). Clone RF113 has sequence similarityto a family of cytokinin-induced transcripts (cig2)that are specifically up-regulated by cytokinins andfunction as GDP/GTP exchange factors (eIF2b) andregulate translation initiation.

Two chloroplast-localized transcripts were clonedby SHMB and found to be significantly up-regulatedon the microarray. The DH104 fragment correspondsto the Arabidopsis biosynthetic enzyme of the thia-mine precursor thiazole (Ribeiro et al., 1996), and theDH054 fragment corresponds to N. tabacum sulfitereductase (Yonekura-Sakakibara et al., 1998). Thia-mine functions as cofactor for two enzyme complexesof pyruvate dehydrogenase and alpha-ketoglutaratedehydrogenase in the citric acid cycle (Belanger et al.,1995). Sulfite reductase catalyzes the six electron re-ductions of sulfite to sulfide and nitrite to ammoniausing electrons donated from ferredoxin.

One of the more strongly up-regulated transcriptsby M. sexta attack was a DH193, which has similarityto an MIP2 from potato (G. Leggewie, L. Willmitzer,and J.W. Riesmeier, unpublished data). MIPs are asuperfamily of membrane channel proteins, some ofwhich are known to function as aquaporins or wateror neutral solute facilitators and tend to be inducedby water or salt stress in various Nicotiana spp.(Yamada et al., 1997; Smart et al., 2001).

Given that M. sexta attack results in large-scaleremodeling of metabolism, perhaps it is not surpris-ing to find several genes for DNA- and RNA-bindingproteins, RNPs, and ribosomal RNA, which togethersuggest a remodeling of the transcriptional machin-ery. DH017, which tended to be up-regulated, hassimilarity to an N. tabacum matrix attachmentregions-binding protein (M. Maeshima and S. Fuji-wara, unpublished data), which is thought to playmultifunctional roles in chromatin organization andmay control the accessibility of promoters to factorsrequired for transcription (Hatton and Gray, 1999).Similarly, DH219, which has similarity to the H3class of histones (S.C. Wu, P. Gregersen, and K. Hahl-brock, unpublished data), proteins known to orga-

Hui et al.


nize chromatin and nucleosome structure and influ-ence the fundamental nuclear processes oftranscription, replication, and DNA repair, wasstrongly up-regulated by M. sexta attack. RN161 wassignificantly up-regulated and has similarity to thetransformer-2-like protein (Petitot et al., 1997) that isa Ser-/Arg-rich RNP family thought to play a impor-tant role in the regulation of constitutive and alter-native splicing of nuclear pre-mRNA. A similar func-tion has been attributed to DH162, a transcript withsimilarity to the N. sylvestris RNA-binding Gly-richprotein (RGP-1a; Hirose et al., 1993). Both of theseputative RNA-binding transcripts exhibited Type Iaexpression patterns, which increased steadily as her-bivore attack progressed. Two ribosomal RNA genes(DH114 and RB493) were found to show differentexpression patterns in the time course. The internaltranscribed spacer of 26S ribosomal RNA gene(DH114) was significantly down-regulated at the 2-hharvest, whereas RB493, which had similarity to Z.mobilis rrnB operon and 23S ribosomal RNA genes,was significantly up-regulated at the same harvest.RD131, with similarity to the 7S RNA RNP complexof signal recognition particles (Riedel et al., 1995) thatmediates the targeting of proteins to the endoplasmicreticulum, was not significantly regulated at any har-vest but tended to increase with herbivore attackfrom an initial down-regulated state. Interpretationsof differential regulation of ribosomal RNA specieswith microarrays that use cDNAs reverse transcribedfrom mRNA species as probes should be viewed withcaution because it is unknown whether the ribosomalRNA is quantitatively amplified.

DISCUSSION

Experimental Approach

We cloned 115 transcripts from the insect-responsive transcriptome of N. attenuata by SHMBand DDRT-PCR using six of the possible 26 arbitraryprimers (Liang et al., 1993) thereby extending theinitial DDRT-PCR analysis of this plant-insect inter-action (Hermsmeier et al., 2001) from approximately4% to 26% of the herbivore-induced transcriptome.The proportion of the transcriptome covered by theSHMB analysis is more difficult to estimate, butgiven that no gene was cloned by both procedures, alarge number of genes were probably involved. BothSHMB and the DDRT-PCR, unlike other fingerprint-ing techniques, provide an unbiased view of the tran-scriptional changes elicited during a plant-insect in-teraction. These two procedures, however, differ intheir ability to simultaneously detect transcripts thatare induced and repressed by the interaction. OnlyDDRT-PCR allows for this possibility in a single ex-periment. This advantage of DDRT-PCR is balancedby the higher proportion (76% versus 45%) of cloneswithout significant similarities to known genes inBLAST queries, a distinct disadvantage of a proce-

dure that utilizes poly(A�)-rich anchor primers and,as a consequence, delivers sequence from the 3�-untranslated region of induced genes (Appel et al.,1999). This disadvantage will presumably decrease asthe number of sequences available in the databasesincreases. An additional difficulty of DDRT-PCR isthe high rate of apparent false-positives and thelabor-intensive verification procedures for detectingdifferential expressions (Appel et al., 1999). More-over, the most commonly used verification proce-dure, the northern blot, may not be sufficiently sen-sitive to detect differential expression in rarelyexpressed transcripts: a putative advantage of theDDRT-PCR procedure.

To minimize the labor associated with verification,the clones were arrayed as cDNAs, and differentialexpression (arbitrarily defined as having an expres-sion ratio of �0.5 or �1.50) was verified for 73 clones,with approximately equivalent proportions being de-rived from the SHMB and DDRT-PCR procedures.Hence, by these criteria, the rate of false-positives didnot differ between the two display procedures. EightN. attenuata genes, whose transcriptional responsesafter M. sexta attack had been characterized previ-ously by northern-blot analysis, were included on themicroarray, and in all cases, their expression ratioswere consistent with the patterns observed in thenorthern analyses. The microarrays not only allowedfor the verification of differential expression in plantsthat were under continuous attack for 24 h, the timewhen the SHMB and DDRT-PCR analyses were per-formed, but by analyzing expression patterns after 2,6, and 12 h of continuous attack, they documentedthe ontogeny of the differential expression patterns.In some cases, these expression patterns suggestfunctional associations between previously unassoci-ated genes. This approach yielded a number of in-sights into the transcriptional changes that occur dur-ing the interaction. However, it should be noted thatthese responses require confirmation by northern-blot analysis.

Oxylipin Signaling

JA elicitation of N. attenuata is known to conferdramatic induced resistance in both field (Baldwin,1998) as well as laboratory (van Dam et al., 2000,2001a) trials with M. sexta larvae. Moreover, M. sextaattack is known to result in a JA burst and increasesin NaAOS transcripts (Ziegler et al., 2001). The coor-dinated increases in LOX, AOS, and HPL transcriptsobserved in this study (Fig. 2) are consistent withthose reported from other species (Reymond et al.,2000; Sasaki et al., 2001) and are correlated with M.sexta attack-induced changes in JA and C6 volatilesbut are too slow to account for their induced changesin metabolites (Kessler and Baldwin, 2001; Ziegler etal., 2001). Although the importance of these genes inplant-herbivore interactions is being convincingly



demonstrated with plants deficient in their expres-sion (for review, see Blee, 2002), the function of thetranscriptional changes remains enigmatic.

The microarray analysis provided evidence for thesimultaneous activation of SA- (DH099), ethylene-(PI), cytokinin- (RF113), and JA (Fig. 2)-signalingpathways during massive herbivore attack. The co-activation of numerous signal cascades in response tovarious biotic and abiotic stresses has been found innumerous studies using Arabidopsis microarrays(Maleck et al., 2000; Schenk et al., 2000; Moran andThompson, 2001; Sasaki et al., 2001; Chen et al., 2002)and suggests that what had been described previ-ously as linear signal cascades associated with par-ticular elicitors are in fact a network of interactingcascades. Moreover, the inhibition of the JA cascadeby the SA cascade may not be occurring during cat-erpillar attack as suggested by the strong up-regulation of transcripts for PR-1 (Fig. 3), a hallmarksignature of SA signaling (Payne et al., 1989), inconjunction with those for PMT and PIs, signaturesof JA signaling (Van Dam et al., 2001b; Winz andBaldwin, 2001).

An additional intriguing correspondence was ob-served between expression patterns of the oxylipinbiosynthetic genes and those of clones RN254 andRC095, which had similarity to the tomato ubiquitin-conjugating enzyme (E2). The JA-insensitive coi1 mu-tant of Arabidopsis, which is defective in most JA-mediated defense signaling, has recently been shownto result from a single amino acid change in the F-boxmotif of the COI protein and abolishes the formationof a ubiquitin-ligase complex (Xu et al., 2002). In theubiquitin-dependent proteolytic pathway, ubiquitinis linked to particular substrates to activate targetingvia the sequential actions of a ubiquitin-activatingenzyme (E1), a ubiquitin-conjugating enzyme (E2),and a ubiquitin ligase (E3). Although the targeting ofparticular proteins for degradation is thought to bemediated by the E3 complex, the other two are es-sential for the function of the complex, and it isunclear which elements may be limiting during pe-riods of large metabolic reconfigurations. AnotherF-box protein within the E3 complex has been re-ported recently to mediate a novel form of SA-mediated pathogen resistance in the Arabidopsisson1 mutant (Kim and Delaney, 2002), suggestingthat both JA- and SA-mediated signaling involves thespecific degradation of particular proteins.

Defense Responses

The “ask the plant” experimental approach used inthis study provided transcripts that had not beenassociated previously with herbivore attack and newinsights into previously characterized M. sexta- andMeJA-induced transcripts. The microarray analysissuggested dramatic increases in PI transcripts andmore modest increases in PMT transcripts (Fig. 1),

consistent with previous work (Baldwin, 2001; VanDam et al., 2001b; Glawe et al., 2003). Interestingly,the down-regulation of the wound-induced increasein PMT transcripts and nicotine accumulation, whichresults from an ethylene burst that is produced whensingle caterpillars or their oral secretions are appliedto mechanical wounds (Winz and Baldwin, 2001),evolves into a sustained increase when plants aremassively attacked by larvae (Fig. 1). The down-regulation of the nicotine response is thought to bean optimization of defense responses because theplant switches from using a metabolically demand-ing direct defense, which could be coopted by anicotine-tolerant herbivore for its own defensive pur-poses, to a metabolically inexpensive but effectiveindirect defense (Baldwin, 2001). Because this analy-sis did not include plants that had suffered the sameamount of damage but were not exposed tocaterpillar-derived signals, it is difficult to determinethe degree to which the nicotine response was down-regulated. However, it is clear that the down-regulation was not complete, suggesting that duringmassive herbivore attack plants can readjust theirdefense responses if, for example, the loss of theirentire canopy is imminent.

In addition to providing new kinetic informationon previously characterized transcripts, the analysissuggested that previously uncharacterized tran-scripts underpin responses that had been phenotyp-ically characterized. The dramatic up-regulation ofHMGR and the more modest up-regulation of EASmay reflect the metabolic commitment to terpenoid-based indirect defenses, which are demonstrably ef-fective in nature for N. attenuata (Kessler and Bald-win, 2001). In addition, the analysis provided anumber of transcripts (PR1, metalothionein, PTO,and ABC transporters) that have been associatedwith defense against microbes. The up-regulation ofthese transcripts suggests that more attention shouldbe given to the direct effect of these defensive pro-teins on insect herbivores or their potential indirecteffects by inhibiting the microbial endosymbiontsfrequently found in insect herbivores.

The most frequently sequenced clones from theDDRT-PCR were the two thigmomorphogenticallyresponsive clones with similarity to XTHs, whoseexpression increased (3.44-fold) during massive her-bivore attack with a Type IV expression pattern (Fig.3). Reducing XTH enzyme activity is thought tostrengthen cell walls (Herbers et al., 2001) becausethis enzyme cuts XG polymers and inserts glucansubunits into existing cell wall polymers. The result-ing average length of the XGs in the cell walls de-creases. Decreasing the number of long linear cellwall polymers is thought to diminish the strength ofcell wall fibers, so it is unlikely that the herbivore-induced increase represents a strengthening of cellwalls as a defensive response against herbivore at-tack (Herbers et al., 2001; but see Braam et al., 1996).

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Cell walls represent a source of carbohydrate-basedelicitors, and if XTH transfers an XG to water or otherXGs not linked to the matrix of the cell wall, thesetranscripts might conceivably influence defensivesignaling in a manner analogous to the cev1 mutantof Arabidopsis. Cev1 is defective in cellulose syn-thase regulation and exhibits constitutive expressionof ethylene and JA response genes (Ellis et al., 2002).The extracellular matrix of plant cell walls clearlyrepresents a rich source of paracrine elicitors (Brown-lee, 2002); XTH up-regulation may play a role ingenerating these elicitors. Alternatively, XTH may beinvolved in loosening cell walls (Braam et al., 1996) inresponse to herbivore attack. By allowing plants toalter their shape and potentially speed the recoveryof the photosynthetic canopy lost to herbivores, XTHmay increase a plant’s tolerance to herbivore attack.

Tolerance and Other “Civilian” Responses to Herbivory

Plant responses that decrease the amount of tissuelost to herbivores (defensive traits) are only onemeans of minimizing the fitness consequences of her-bivore attack. Responses that minimize the fitnessconsequences of losing tissue to herbivores (toleranceresponses) represent an equally effective but largelyunstudied means of coping with herbivore attack.Mobilizing limiting resources from tissues that areabout to be consumed into parts that are less likely tobe eaten by herbivores (petioles, stems, and below-ground tissues) may represent such a mechanism.Such mobilization may share components with themetabolic mobilization of resources required for theproduction of defense traits. The resources requiredfor the resistance traits could be made available bydecreases in the five photosynthetic-related tran-scripts, increases in transcripts associated with pro-tein, and nucleotide turnover and increases in tran-scripts associated with carbohydrate metabolism.Whether or not these transcriptional changes play arole in balancing the plant’s resource budgets re-mains to be determined.

CONCLUSION

M. sexta attack results in a large-scale transcrip-tional changes in N. attenuata genes that are collec-tively consistent with a reconfiguration of metabo-lism that reduces photosynthetic activity, slowsgrowth, and increases a diversity of defense traits.Numerous signal cascades appear to be involved incoordinating the responses. These coordinatedchanges point to the existence of central herbivore-activated regulators of metabolism, which in turn areactivated by minute amounts of FACs in M. sexta’soral secretions (Schittko et al., 2000, 2001; Halitschkeet al., 2001). In a companion paper, we use microar-rays to examine the proportion of the M. sexta-induced transcriptome that is elicited by FACs.

MATERIALS AND METHODS

Plant Growth

An inbred line of Nicotiana attenuata Torr. ex Wats. originally collectedfrom southwestern Utah in 1988 was used for all experiments and was thesame genotype used by Hermsmeier et al. (2001). Seeds were germinated inpotting soil after soaking with a 1:50 (w/v) dilution of liquid smoke (Houseof Herbs, Passaic, NY). One-week-old seedlings were transferred to 28-Lcommunal hydroponic boxes with a nutrient solution consisting of 0.292 gL�1 of Peter’s Hydrosol (W.R. Grace, Fogelsville, PA) and 0.193 g L�1 ofCa(NO3)2. After an adaptation period of 5 d, seedlings were transferred toindividual 1-L hydroponic chambers containing a no-nitrogen hydroponicsolution (Baldwin et al., 1994). Nitrogen was added after transfer by adding2 mL of a 1 m KNO3 to each chamber and 1 mL a day before placing larvaeon plants. Plants were placed in a growth chamber with a photoperiodiccycle programmed for a 16-h light period at 32°C and an 8-h dark period at28°C with 65% constant relative humidity. Forty of the most similar lookingplants in the rosette stage of growth were chosen for the display experi-ments, and in a separate experiment, 80 plants were chosen for the microar-ray experiment.

Insect Rearing and Plant Treatments

The eggs of Manduca sexta (Lepidoptera, Sphingidae) from CarolinaBiological Supply (Burlington, NC) were hatched at 28°C. For the displayexperiment, 20 first instar larvae were placed on each of 20 plants at 12 pm(6 h into the light cycle), with one to three larvae per leaf, depending on theleaf size. After 24 h of feeding, the larvae and frass were removed, and 20attacked and 20 control plants were harvested, separated into shoots androots, immediately placed in liquid nitrogen, and stored at �80°C until usedfor DDRT-PCR and SHMB. Plants used in the microarray experiment weregrown and treated identically as those used in the display experiment,except that 10 attacked and 10 control plants were harvested 2, 6, 12, and24 h, respectively, after larvae were placed on plants.

DDRT-PCR

Procedures follow closely those described in Hermsmeier et al. (2001)with minor modifications. Total RNAs of shoots and roots were extractedseparately from 5-g aliquots of 20 attacked and control plants. GenomicDNA was removed by adding 20 units of RNase free DNase I (Life Tech-nologies, Eggenstein, Germany) for each 100-�L reaction volume containing100 �g of total RNA. DNA-free RNAs were adjusted to a 1:1 (root:shoot)concentration for each reverse transcription (RT). First strand cDNAs weresynthesized with 400 ng of purified total RNAs and 25 �m anchor primersA1(T12AA), A2(T12AC), A3(T12AG) A4(T12CA), A5(T12 CC), A6(T12CG),A7(T12GA), A8(T12GC), A9(T12GG), and A10(T12GT) (MWG Biotech, Mu-nich), 200 units of SuperScript-II reverse transcriptase (Life Technologies),and 200 �m dNTPs, respectively. The reactions of each anchor primerthat did not receive reverse transcriptase served as quality controls forpotential RNA contamination by residual genomic DNA, which was lateramplified in the DDRT-PCR procedure. DDRT-PCR was performed with eachRT reaction with Platinum Taq polymerase (Life Technologies), dNTPs in-cluding �-33P labeled dCTP (NEN Life Science, Zaventem, Belgium), arbitraryprimers R2(TGGATTGGTC), R3(CTTTCTACCC), R4(TTTTGGCTCC),R5(GGAACCAATC), R6(AAACTCCGTC), and R14(GATCAAGTCC) in com-bination with anchor primers A1 to A10, respectively. Thermocycling param-eters were: denaturation at 94°C for 2 min for activation, followed by 40 cyclesof 30 s of denaturation at 94°C, 120 s of annealing at 40°C, and 30s of extensionat 72°C. The PCR amplification products were separated on a 6% (w/v)polyacrylamide denaturing gel. Gels were dried on Whatman 3MM paper(Whatman, Clifton, NJ) and exposed to Kodak Biomax MR film (AmershamPharmacia Biotech, Freiburg, Germany). The developed films were used astemplates to excise the differential (both amplified and suppressed) bandsfrom the display gel. The cDNAs were eluted from the gel by incubating gelslices in 150 �L of water for 10 min at 25°C, 15 min at 100°C, then transferringto 4°C, followed by centrifugation at 13,000 rpm for 2 min, and the cDNAswere recovered from the supernatant. To clone the eluted cDNA fragments,the TOPO TA cloning kit (Invitrogen, CH Groningen, The Netherlands) wasemployed directly with the PCR products that were re-amplified by usingcorresponding primers and the PCR temperature program given above. Plas-



mid DNA was isolated with NucleoSpin plasmid kit (Macherey-Nagel, Du-ren, Germany) for sequencing. Plasmid inserts were sequenced on an ABIPrism 377 XL DNA sequencer with the Big Dye terminator kit (PE-AppliedBiosystems, Weiterstadt, Germany) and analyzed with the Lasergene soft-ware package (DNASTAR, Madison WI).

SHMB followed closely the protocol of Sharma et al. (1993) using Dyna-beads as described in “Method 1” of the manufacturer’s instructions (DynalBiotech, Hamburg, Germany). The mRNAs from attacked (tester) and con-trol (driver) plants were isolated with paramagnetic oligo(dT)25 beads(Dynabeads) from 100 �g of total RNAs treated with DNase I (Life Tech-nologies). Driver mRNA on the beads was directly converted to the com-plementary first strand cDNA using SuperScript-II reverse transcriptase andrTth reverse polymerase (Life Technologies) according to the manufactur-er’s instructions. Tester mRNA was then eluted from the beads and hybrid-ized to the driver cDNA, which was immobilized on the Dynabeads by theRT reaction. After three stringent hybridizations (each for 24 h) in 4.5� SSPEand 0.1% (w/v) SDS buffers at 68°C and removal of the subtracted mRNA,20 �L of fresh oligo(dT)25 beads was used for collecting the mRNA left aftersubtraction. The eluted mRNA was reverse transcribed to the first strandcDNA using SuperScript-II reverse transcriptase (Life Technologies) andoligo(dT)21 (MWG Biotech). Subsequently, DNA polymerase I and RNase Hwere used for second strand synthesis. Double-strand cDNA was bluntended with T4 DNA polymerase (Amersham Pharmacia Biotech) and sub-sequently treated with T4 polynucleotide kinase for cloning to pUC18 vectorprepared by restriction with SmaI enzyme.

Fabrication of cDNA Microarray. Fabrication ofcDNA Microarray

The cDNAs cloned in the pCR2.1-TOPO (Hermsmeier et al., 2001) andpUC18 vectors were PCR amplified with the following primers derived fromvector sequences close to the insert: TOP5-20, 5�-CAGTGTGCTGGAAT-TCGCCC-3�; TOP6-21, 5�-GGATATCTGCAGAATTCGCCC-3�; SMA1-19, 5�-GAATTCGAGCTCGGTACCC-3�; SMA4-23, 5�-CAGGTCGACTCTAGAG-GATCCCC-3�; SMA3-22, 5�-TACGAATTCGAGCTCGGTACCC-3�; andSMA2-20, 5�-GTCGACTCTAGAGGATCCCC-3�. For pCR2.1-TOPO, TOP5-20, and TOP6-21 were used. For pUC18, primer pairs SMA3-22 and SMA2-20and SMA1-19 and SMA4-23 were used.

For the preparation of the well-characterized control genes, plasmidpNATGUS3 (Krugel et al., 2002) digested with BstEII and NcoI was used as avector to clone the following N. attenuata gene PCR fragments digested withthe same enzymes: pi, primers, PIA1-34 (5�-GCGGCGGGTCACCGTACTTTA-GTGATGATGGAAC-3�) and PIA2-32 (5�-GCGGCGCCATGGCTTACAAC-CCTTCGTGCCTG-3�); template, chromosomal DNA of N. attenuata; pmt1,primers, PMT6-36 (5�-GCGGCGGGTCACCGGTACCAACACAAATGGC-TCTAC-3�) and PMT7-31 (5�-GCGGCGCCATGGAGCCCTTAAAGACTT-GACG-3�); template, pmt1 cDNA cloned on plasmid pBI121-ASPMT (Voelckelet al., 2001); aos, primers, AOS1-35 (5�-GCGGCGGGTCACCGTGTTCTTTCT-TATCTTGATCC-3�) and AOS2-31 (5�-GCGGCGCCATGGAAGTAG-GAAAACCAAGAAC-3�); template, chromosomal DNA of N. attenuata; xet,primers, XET1-32 (5�-GCGGCGGGTCACCATTCACAGCTTCTTACAGG-3�)and XET2-33 5�-GCGGCGCCATGGCCTTGAACGCTTGCATTCAGG-3�);template, RB271 (this publication); and wrky, primers, TFN1-34 (5�-GCGGCGGGTCACCGGAACCAATCATGGAATTATC-3�) and TFN2-31 5�-GCGGCGCCATGGTGGGACAATTTGGGAAAG-3�); template, RE322 (thispublication), yielding plasmids pNATPI1, pNATPMT1, pNATAOS1,pNATXET1, and (with wrky fragment) pNATTFN1, respectively. Afterward,the N. attenuata control gene PCR products for spotting onto the chip weresynthesized as follows: pi, hpl, pmt1, aos, xet, and wrky with primers ASV5-21(5�-GGAGAAACTCGACCGGTCACC-3�) and ASV6-22 (5�-CTACAAATC-TATCTCTCCATGG-3�); templates pNATPI1, pNATHPL1 (Krugel et al.,2002), pNATPMT1, pNATAOS1, pNATXET1, and pNATTFN1, respectively;3� region of lox with primers LOX4-22 (5�-CTTTGGCGTTTTGATTTGGAAG-3�), ASV6-22, template pNATLOX1 (Krugel et al., 2002), and 5� region of loxwith primers ASV5-21, LOX3-21 (5�-CCAGTGCGACAACGTCTTGGG-3�),and template pNATLOX1. For each cDNA, two PCR fragments, with 5�-Aminolink C6 modification (Sigma-ARK, Darmstadt, Germany) on eitherstrand, were synthesized. Even-numbered fragments (Table I) carry theAminolink modification at primers TOP5-20, SMA4-23, or ASV6-22, whereasodd numbered fragments carry the modification at primers TOP6-21, SMA3-22, or ASV5-21.

PCR products were purified by a PCR purification kit (QIAquick, Qiagen,Hilden, Germany) following the manufacturer’s instructions. Agarose gelelectrophoresis was performed to confirm the purity, and the concentrationof the amplified products was determined spectrophotometrically. Com-mercially available Lys-coated slides (PL-25C Poly-l-Lys slides, CEL Asso-ciates, Inc., Houston) were used. Before spotting, all the cDNA samples wereconcentrated through a micron-MultiScreen-PCR (Millipore, Bedford, MA)to approximately 0.5 to 1.0 �g �L�1 with spotting solution from Telechem(CEL Associates).

All the cDNA samples were arrayed four times (so that each gene wasrepresented by eight spots) on the slides by a robot equipped with fourprinting tips (OmniGrid Microarrayer, Genemachine, San Carlos, CA). A listof genes on the microarray is in Table I. The spotted DNA on slides washydrated in 1� SSC buffer for 1 to 5 min and snap dried at 140°C for 3 s,followed by cross-linking with a Stratalinker-2400 apparatus (Stratagene, LaJolla, CA). To prevent intrinsic fluorescence, the surface of the slides wastreated with a blocking solution containing 5.5 g of succinic anhydridedissolved in 335 mL of 1-methyl-2-pyrrlidinone, mixed in 15 mL of 1 mNaBorate prepared with boric acid, and adjusted with NaOH to pH 8.0.Finally, spotted DNA on slides was denatured in boiling water for 2 min,rinsed with ethanol, and dried by centrifugation. After prehybridizationprocessing, sample slides were hybridized with Cy3- or Cy5-labeled randomprimers (9 mer) to examine qualitative characteristics of the microarrays.

Microarray hybridization and quantification: Poly(A�) RNAs were iso-lated from 100 �g of total RNA (adjusted to a 1:1 [root:shoot] concentration)with Dynabeads Oligo(dT)25 (Dynal Biotech) and used for RT. To synthesizethe first strand, 2 �g of poly(A�) RNAs was mixed with 4 �g of randomhexamer (pdN6, Sigma), 4 �g oligo(dT)22 (Sigma) in 15 �L, and incubated at65°C for 10 min. Subsequently, 0.6 �L of 50� dUTP/dNTPs [10 �L of each100 mm dATP, dGTP, and dCTP; 6 �L of 100 mm dTTP; and 4 �L of 100 mmdUTP [5-(3-Aminoallyl)-2�-deoxyuridine 5�-triphosphate sodium salt, Sig-ma], 6 �L of 5� buffer, 3 �L of dithiothreitol (0.1 m), 1.9 �L of SuperScript(RNase II Hfree) reverse transcriptase, and 3.5 �L of water were added to avolume of 30 �L and incubated at 42°C for 2 h. cDNA/mRNA hybrids werehydrolyzed with 10 �L of NaOH (1 n) and 10 �L of EDTA (0.5 m) andincubated at 65°C for 15 min after neutralization with 25 �L (1 m) Tris(pH 7.4).

The cDNA mixtures were cleaned with a Microcon 30 concentrator(YM-30, Millipore) and dried in a speed vac. The pellets of both induced andcontrol sample were resuspended in 9 �L of NaHCO3 buffer (0.5 m, pH 9.0)and added to the dried aliquot of monofunctional NHS-ester Cy3 dye and toCy5 dye (Amersham Pharmacia Biotech), respectively, for labeling at roomtemperature in darkness. After 1.5 h, the Cy3 and Cy5 reactions werequenched with 4.5 �L of hydroxylamine (4 m) and mixed. After purificationwith Qiaquick PCR purification kit (Qiagen), the eluted products were driedin a speed vac. The labeling efficiency of the cDNA probe was checked bya spectrophotometer at a wavelength of 200 to 700 nm.

The probe solution was prepared by resuspending the dried pellets in ahybridization buffer consisting of 2 �L of poly(A�)(22) (10 �g �L�1, MWGBiotech), 5 �L of 20� SSC, 2 �L of yeast-tRNA (1.25 �g �L�1, Life Tech-nologies), 0.6 �L of 10% (w/v) SDS, and 20.4 �L of distilled water for a finalvolume of 30 �L. The probe solution (after heating at 95°C for 2 min) washybridized to the microarray, which was denatured in boiling water for 2min, dried before use, and covered with a silanized coverslip. Hybridizationwas carried out for 12 h in a hybridization chamber (a 50-mL falcon tubesupplied with 2 mL of 20� SSC on Whatman paper) and placed in ahybridization oven at 55°C to 58°C. After hybridization, the slides wereimmediately washed, initially with a solution of 1� SSC and 0.1% (w/v)SDS for 15 min, then with a solution of 1� SSC for 5 min, before being driedby centrifugation (3 min at 1,000 rpm).

A ScannArray-3000 (GSI Lumonics, Watertown, MA) was used to scanthe hybridized cDNA with sequential scanning for Cy5 cDNA and then forCy3cDNA at a maximum resolution of 10 �m pixel�1 with a 16-bit depth.The hybridization images were evaluated using the program AIDA ImageAnalyzer (Raytest Isotopenme�grate GmbH, Straubenhardt, Germany).Each image was overlaid with a grid to assess the signal strength from eachspot. The background correction was manually calculated around each spotwith a depth of 2 pixels. To calculate a microarray-specific normalizationfactor, the measured Cy5 and Cy3 fluorescence intensities were rankedindependently and after discarding the 12.5% maximum and minimumvalues, the remaining 75% of the values were summed. The array-specificnormalization factor was obtained by dividing the calculated sum of Cy3values by those of the Cy5 values. The ratios of normalized fluorescence

Hui et al.


values for Cy3 and Cy5 of each individual spot for which an adequatebackground correction could be determined (typically five–six of the eightreplicate spots for each gene) were used to calculate the mean and (sds) foreach cDNA. We arbitrarily defined cDNAs with mean expression ratios of�0.5 and �1.50 as being differentially expressed (down- and up-regulated,respectively).

ACKNOWLEDGMENTS

We thank Susan Kutschbach for the DNA preparation; Thomas Hahn andDominika Schnabelrauch for sequencing; Evelyn Claussen for assistancewith the figures; Anja Paschold, Kristine Brathen, and Katja Schenke fortechnical assistance; and Rayko Halitschke, Claudia Voelckel, and EmilyWheeler for helpful comments on the manuscript.

Received November 22, 2002; returned for revision December 26, 2002;accepted January 14, 2003.

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